Abstract

Fibroblast growth factor-1 (FGF-1) is a well characterized growth factor among the 22 members of the FGF superfamily in humans. It binds to all the four known FGF receptors and regulates a plethora of functions including cell growth, proliferation, migration, differentiation, and survival in different cell types. FGF-1 is involved in the regulation of diverse physiological processes such as development, angiogenesis, wound healing, adipogenesis, and neurogenesis. Deregulation of FGF-1 signaling is not only implicated in tumorigenesis but also is associated with tumor invasion and metastasis. Given the biomedical significance of FGFs and the fact that individual FGFs have different roles in diverse physiological processes, the analysis of signaling pathways induced by the binding of specific FGFs to their cognate receptors demands more focused efforts. Currently, there are no resources in the public domain that facilitate the analysis of signaling pathways induced by individual FGFs in the FGF/FGFR signaling system. Towards this, we have developed a resource of signaling reactions triggered by FGF-1/FGFR system in various cell types/tissues. The pathway data and the reaction map are made available for download in different community standard data exchange formats through NetPath and NetSlim signaling pathway resources.

1. Introduction

Fibroblast growth factor (FGF) superfamily consists of structurally related polypeptides most of which function through its high affinity fibroblast growth factor receptors (FGFRs). In addition to FGFRs, they also bind to heparan sulfate proteoglycans (HPSGs) and their analog, heparin. These interactions influence the stability of FGFs in the extracellular matrix and also regulate their binding and activation of FGFRs [1–9]. In humans, FGFs are encoded by 22 genes, FGF-1-14 and FGF-16-23, and are divided into 7 subfamilies. FGFs 1–10 and 16–23 are FGFR ligands, while FGFs 11–14 are intracellular FGF homologous factors which act in a receptor-independent fashion [10]. Knock-out mice of different FGFs exhibit diverse developmental and physiological disorders [11]. For instance, FGF-9 is involved in the development of lung and testes [12, 13], FGF-3 is critical for inner ear development [14], and FGF-18 is important in bone and lung development [15–17]. Moreover, knock-out of FGFs 4, 8, 9, 10, 15, 18, or 23 was found to be lethal in mice [18]. FGFs are also involved in wound healing, tissue repair [19, 20], and angiogenesis [21]. Facilitating cell proliferation, migration, and differentiation [16, 22–26], FGFs are implicated in diverse pathological conditions including cancer [27] as well as metabolic and developmental disorders [18].

Most FGFs have an N-terminal signal peptide and are thus secreted. FGFs 1, 2, 9, 16, and 20 do not have signal peptides. FGFs 9, 16, and 20 may be released through classical secretory pathway; however, FGF-1 and FGF-2 are released from damaged cells or through endoplasmic reticulum-golgi independent exocytotic pathway [10]. FGF-1 along with FGF-2 was initially isolated from bovine pituitary extracts based on their ability to induce proliferation in 3T3 fibroblasts [28, 29]. Also known as acidic FGF, FGF-1 is a 155 amino acid long non-glycosylated polypeptide. FGF-1 is not released from the cells under normal physiological conditions, but it was secreted in response to stress conditions such as heat shock, hypoxia [30, 31], serum starvation [32], and exposure to low-density lipoproteins [33]. Stress induces the release of inactive disulfide bond-linked homodimeric form of FGF-1, which is dependent on p40-Syt1, S100A13, and Cu2+ ions [34–37]. FGF-1 has been shown to reduce apoptosis in vascular injury [38–40]. Administration of FGF-1 has shown promise as a therapeutic strategy against human cervical spinal cord injury [41] and ischemic conditions [42–44]. Increased expression of FGF-1 was observed in ovarian [45] and prostate cancers [46]. Taken together, FGF1 is involved in different cellular functions that are mediated through its interaction with the four FGF receptors [47, 48]. A pathway resource representing these diverse functions and the underlying mechanisms that regulate these processes would be immensely useful.

Curated pathway maps are invaluable resources for scientific community. Such comprehensive pathway datasets are being increasingly used in bioinformatics efforts directed towards analysis of high-throughput datasets from various disease contexts. Repositories including Pathway Interaction Database of the National Cancer Institute (http://pid.nci.nih.gov/), Database of Cell Signaling (http://stke.sciencemag.org/cm/), KEGG Pathway Database (http://www.genome.jp/kegg/pathway.html), and INOH Pathway Database (http://inoh.org/) have cataloged basic components of FGF signaling. We have expanded the scope of this by providing a comprehensive representation of FGF1 signaling pathway and its diverse roles in regulating various cellular processes.

2. Methodology

Documentation of specific pathway reactions scattered in the literature into an organized, user-friendly, query-enabled platform is primary to the analysis of signaling pathways. We used NCBI PubMed database to carry out an extensive literature search to retrieve research articles where molecular events triggered by the FGF-1/FGFR signaling system were studied. Specific molecular events screened include (a) physical associations between proteins, (b) posttranslational modifications (PTMs), (c) change in subcellular localization of proteins, (d) activation or inhibition of specific proteins, and (e) regulation of gene expression. Relevant information from research articles were manually documented using the curation tool, PathBuilder. To streamline and organize data collection from literature, we followed the previously described criteria for the inclusion/exclusion of pathway specific reactions [49, 50]. The data accumulated was submitted to the NetPath signaling pathway resource developed by our group [51]. We then generated a signaling map for this pathway using PathVisio pathway visualization software. We also applied additional criteria to filter out low confidence reactions from the gathered data [52] and generated a NetSlim map. In addition to curation of molecular level information, we have also cataloged physiological effects brought about by FGF-1 in different cell types/tissues.

3. Results and Discussion

Canonical FGF/FGFR signaling reactions have been documented in a few public repositories and review articles. Vast amount of literature in the last few years have revealed several novel pathway intermediates of FGF/FGFR signaling system. In order to generate a comprehensive view of FGF/FGFR signaling pathway, we carried out extensive literature search on PubMed for articles pertaining to FGF-1 signaling. Of a total of 3275 articles that were screened, 237 of them had molecular reactions reported downstream of FGF-1 in various cell types/tissues. Manual curation from these research articles revealed 109 molecules involved in FGF-1 induced physical associations, modulation by PTMs, activity, and subcellular or cell surface translocation events. Of the 42 physical associations that were cataloged, 29 were “binary” and 13 were “complex” interactions inclusive of the ligand/receptor interactors. We could record a total of 87 catalysis events, 15 activation/inhibition, and 21 translocation events. The 87 catalysis events include 19 events, where the enzymes directly catalyzing the reactions were studied and reported, and 68 events for which the enzymes which post-translationally modified the proteins are not studied under FGF-1 stimulation. Apart from these molecular reactions, we have also cataloged 117 genes whose expression is reported to be either upregulated or downregulated by FGF-1 treatment. However, only a total of 25 genes were reported to be differentially regulated at mRNA level by FGF-1 stimulation in different human cell types. A list of genes reported to be regulated by FGF-1 in different mammalian systems at the mRNA and/or the protein level is provided in Table 1. After the annotation process, all the entries were reviewed and approved by internal reviewers. Internally reviewed pathways were further reviewed and approved by an external pathway authority (LC, who is an author in this paper).

Table 1: List of genes that are reported to be transcriptionally and translationally regulated by FGF-1 in humans and other mammals.

3.1. Signaling Modules Activated by FGF-1

Signaling modules comprise a well-characterized group of molecules and their interactions downstream of activation of a receptor. We documented the following signaling modules to be activated upon stimulation with FGF-1.

3.1.1. Ras/Raf/Mek/Erk Pathway

The Ras/Raf/Mek/Erk pathway has been implicated in cellular processes including cell growth, proliferation, and migration. Stimulation of different cell types with FGF-1 resulted in the formation of multiple complexes involving FRS2, GAB1, SOS1, PTPN11, SHC1, SH2B1, and GRB2 [53–60]. These complexes are critical to the subsequent activation of Ras [53, 56]. Association of Ras with Raf kinase [53] induces autophosphorylation and activation of Raf. Activation of Raf leads to phosphorylation dependent activation of Map kinases 1/2 (MAP2K1/2) and subsequently Erk2/1 (MAPK1/3) [60–62]. In the context of FGF-1 signaling, this module was reported to be involved in a number of processes including neurogenesis, adipocyte differentiation, cell proliferation, cholesterogenesis, cardioprotection, and tumor invasion and metastasis [62–67].

3.1.2. Pi3k/Akt Pathway

The complexes mentioned above also lead to the activation of Pi3k/Akt pathway, another signaling module that regulates various processes including cell growth, survival, cell proliferation, and cell migration [68]. A number of studies have shown FGF-1 induced phosphorylation of Akt [63, 64, 69]. Pi3k inhibitor-based functional assays also proved the involvement of FGF-1 pathway in diverse physiological conditions including angiogenesis [70], lung development [71], maintenance of neuronal phenotype [72], neuroprotection [73], and ApoE-HDL secretion [69].

3.1.3. Jnk and p38 Mapk Pathway

The c-jun N-terminal kinase (Jnk) pathway is implicated in the regulation of cell cycle, cell survival and apoptosis. FGF-1 stimulates the phosphorylation of p38 Mapk (MAPK14) as well as Jnk1/2 (MAPK8/9). The Jnk1/2 was also found to be crucial to neurogenesis and vascular remodeling [63, 74]. The specific functions of FGF-1 signaling mediated by p38 Mapk include growth arrest, promotion of apoptosis in response to oxidative stress, and formation of actin stress fibers [75–77].

3.1.4. STAT3 and Nf-kb Pathway

FGF-1 also stimulates STATs (STAT1 and STAT3) and Nf-kB signaling modules. FGFR signaling is reported to be regulated through several downstream molecules including JAK2, SRC, SH2B1, MAPK1/3, MAPK8/9, and STAT3. This signaling axis is known to regulate various cellular processes including neurite outgrowth, cell proliferation, and increased cancer cell invasion [78–80]. In addition, FGF-1 is also reported to induce MMP9 expression in mammary adenocarcinoma cells through the Nf-kb pathway [81].

3.2. Physiological Effects Mediated by FGF-1

FGF-1 was found to be involved in a number of biological processes. It is associated with the development of heart [82], lens [83], lung, and liver [84–86]. Its crucial roles in neurogenesis as well as adipogenesis [65, 87, 88] have also been reported. FGF-1 induces growth arrest and differentiation in chondrocytes [89–92]. It is implicated in angiogenesis [93–95] and wound healing [95–99]. Multiple studies have also shown the role of FGF-1 in cardioprotection [99–101] and neuroprotection [22, 102]. FGF-1 also induces migration [103–105] and proliferation [106–108] in different types of cancer cells. It is also involved in the regulation of epithelial-to-mesenchymal transition [109, 110], and tumorigenesis [111] as well as invasion and metastasis [64, 112]. A list of functional effects of FGF-1 studied in different cell types/tissues is provided in Table 2.

Table 2: Functions of FGF-1 identified in diverse cell/tissue types of human and other mammalian origins.

3.3. Pathway Visualization, Data Formats, and Availability

User-friendly visualization of pathways is an important aspect to provide a concise view. A number of tools are available for visualization and analysis of pathway data including Cytoscape [113], ChisioBioPAX Editor (ChiBE) [114], visualization and layout services for BioPAX pathway models (VISIBIOweb) [115], and ingenuity pathway analysis. These tools use pathway and molecular interaction data in different XML-based community standard data exchange formats as input. These standard formats, which include Proteomics Standards Initiative for Molecular Interaction (PSI-MI version 2.5), Biological Pathway eXchange (BioPAX level 3), and Systems Biology Markup Language (SBML version 2.1), enable easy data exchange and interoperability with multiple software. We have provided the annotated pathway data in the standard formats mentioned above. This data can be downloaded and used from NetPath [51], an open source resource for signal transduction pathways developed by our group (http://www.netpath.org/index.html). Additionally, we have drawn a map of FGF-1/FGFR signaling using the data accumulated in NetPath. This network map represents the molecules and their reactions organized by topology and excludes the molecules identified through phosphoproteomics approaches for which topology could not be assigned (Figure 1). The map was manually drawn using freely available software, PathVisio [116]. The topology of the molecules and their reactions in the pathway was arranged based on (i) inhibitor-based assays, (ii) mutation-based assays, (iii) knock-out studies, (iv) prior knowledge of canonical modules, and/or (v) with reference to multiple review articles. Another map, which incorporated high confidence reactions in accordance with NetSlim criteria [52], is submitted to the NetSlim database. These maps can be visualized and downloaded in gpml, GenMAPP, png, and pdf formats from http://www.netpath.org/netslim/FGF-1_pathway.html. Each node in the map is linked to their molecule page in NetPath, thereby to other pathways in NetPath, and to HPRD [117] and RefSeq protein accessions. In the “map with citation” option, the edges connecting the nodes are linked to the corresponding articles in PubMed that report the FGF-1 stimulated reaction(s). Direct reactions are represented by solid edges. Indirect reactions are represented with dashed edges. The edges which represent the protein-protein interactions, enzyme-substrate reactions and translocation events are distinguished by different colors.

Figure 1: Network map of FGF-1 signaling. This map manually drawn using PathVisio [112] represents the reactions induced by FGF-1 through their receptors. Each node represents the molecules and the post-translationally modified states of proteins are also represented. Distinguished by color and continuous/dashed lines, the edges represent the specific information such as protein-protein interactions, enzyme-substrate reactions, reactions mediated through unknown/multiple steps, and protein translocations as provided in the legend. The biological processes that FGF-1 regulates through multiple signaling modules are also represented. A NetSlim [52] version of this map can be obtained from http://www.netpath.org/netslim/FGF-1_pathway.html.

4. Conclusions

Availability of specific ligand-receptor mediated signaling data in community approved formats is crucial to the understanding of proteins and their reactions in diverse biological processes. Analysis of high-throughput data obtained from microarray- and mass spectrometry-based platforms essentially relies on enrichment of biological function or signaling pathways available in databases to obtain insights into their physiological functions. Although some resources have cataloged FGF signaling in general, this is the first attempt to provide a comprehensive view of FGF-1 signaling. This will be extended to other FGF ligands and/or specific FGFRs in the future to facilitate the analysis of differences between different FGFs and/or FGFRs. The pathway information has been made available through NetPath and NetSlim resources in multiple community standard data formats. The FGF-1 signaling pathway data will be periodically updated in NetPath. We have cataloged multiple signaling modules that are activated upon activation of FGFR and their implications in diverse physiological and pathophysiological processes. We believe that the data presented here will boost further research in this area and will help identify novel therapeutically important molecules that could be targeted in pathological conditions involving aberrant FGF-1 signaling.

Abbreviations

S100A13:

S100 calcium binding protein A13

FRS2:

Fibroblast growth factor receptor substrate 2

GAB1:

GRB2-associated binding protein 1

SOS1:

Son of sevenless homolog 1

PTPN11:

Protein tyrosine phosphatase, non-receptor type 11

SHC1:

Src homology 2 domain containing transforming protein 1

GRB2:

Growth factor receptor-bound protein 2

Mapk:

Mitogen activated protein kinase

Pi3k:

Phosphatidylinositide 3-kinase

Akt:

v-akt murine thymoma viral oncogene homolog

HDL:

High density lipoprotein

Jnk:

Jun N-terminal kinase

STAT3:

Signal transducer and activator of transcription 3.

Conflict of Interests

The authors have no conflict of interests.

Authors’ Contribution

Acknowledgments

The authors thank the Department of Biotechnology (DBT), Government of India, for research support to the Institute of Bioinformatics, Bangalore. Shyam Mohan Palapetta is supported by a Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India. Varot K. Sandhya is a recipient of Inspire Fellowship from the Department of Science and Technology (DST), Government of India. Harsha Gowda is a Wellcome Trust/DBT India Alliance Early Career Fellow.

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